PROJECT SUMMARY Our long-term goal is to develop feasible strategies toward improving the efficacy of cardiac stem/progenitor cell (CPC) therapy, which is severely blocked by the cell source with limited contribution to produce new cardiomyocytes, cell aging, and poor donor cell survival. We previously demonstrated that preconditioning human CPCs with cobalt protoporphyrin (CoPP) exhibited significant beneficial effects for improving cell survival and cardiac function. However, singular strategy has shown insufficient benefit for improving the stem cell therapeutic efficiency. Therefore, the overall objective in this application is to test the compositional three small-molecule compounds to enhance the effectiveness of myocardial repair by a new subset of CPCs with low mitochondrial membrane potential, named as ??mlow-hCPCs. Guided by the strong preliminary data, our central hypothesis is that aging ??mlow-hCPCs have compromised cardiac regenerative potential, while rejuvenating aging ??mlow-hCPCs with combined three small-molecule compounds will restore their cardiac regenerative potential in vitro through targeting multiple senescence signal pathways, and enhance the effectiveness of stem cell therapy for ischemic heart disease in an immuno-deficient murine model following myocardial infarction. This hypothesis will be addressed by pursuing three specific aims: we will first test whether the rejuvenation with a composite of three small-molecule compounds (3SMCs) for aging ??mlow- hCPCs will increase their cardiac regenerative capability, on the basis of a) cell senescent phenotype, b) proliferation, c) cell survival, d) cytokine release, and e) endothelial and cardiomyogenic differentiation, through comparing with the vehicle-treated ??mlow-hCPCs, and with ??mlow-hCPCs from pediatric patients (Aim 1); Our Aim 2 is to understand the underlying molecular and cellular mechanisms of rejuvenating aging ??mlow-hCPCs with combined 3SMCs by targeting the senescence-associated signaling pathways. Finally, we will test whether the transplantation of 3SMCs-treated ??mlow-hCPCs will result in greater improvement of a) in vivo hCPC survival, proliferation, and differentiation; b) endogenous mouse cardiac regeneration; and c) cardiac structure and function in an immune-deficient mouse myocardial infarction (MI) model (Aim 3). The approach is innovative, in the applicant?s opinion, because it is expected to set a new research milestone on applying a composite of small-molecule compounds to enhance the therapeutic effectiveness of the novel subset of hCPCs with low mitochondrial membrane potential. The proposed research is significant, since knowledge gained from these studies will provide the essential implications on the strategies to promote the therapeutic effectiveness of ??mlow-hCPCs after transplantation and, therefore, their efficacy in clinical repairing infarcted myocardium for the cell therapy of patients with ischemic heart disease.